Sewage overflows introduce complex microbial populations containing both commensal and pathogenic organisms into rivers and nearshore waters of the Great Lakes, which have more than 500 recreational beaches and serve as the drinking water supply for more than 40 million people. The prevailing fecal pollution detection indicators Escherichia coli (E. coli) and enterococci lack specificity for host source (animal or human) and are generally a poor indication of the presence of disease causing organisms. New alternative indicators would ideally be better correlated to human pathogens and therefore a more reliable indication of human health risk. We have been generating comprehensive microbial community profiles of sewage and sewage contaminated waters in order to identify candidates for alternative indicators of human fecal pollution. We have generated high-resolution profiles of microbial communities and have identified potential key indicator taxa including members of Lachnospiraceae and Ruminococcaceae. Community profiles of untreated sewage from a single city demonstrate the human taxa signature within these profiles is stable over time and corresponds closely to profiles from other studies of the human intestine. In this work, we will expand our efforts to include geographical comparisons of sewage profiles across 20 locations within the US. We will also extend our work on validating potential alternative indicators by creating profiles of contaminated water samples and utilizing quantitative PCR (qPCR) to establish statistically significant relationships between key taxa within our profiles and pathogenic viruses. The proposed expansion of our efforts is timely because we can accelerate discovery of new alternative indicators by drawing upon data being generated as part of independently funded concurrent studies. Ultimately, alternative indicators may be used within a risk assessment framework to evaluate human health risk from environmental sources of pathogens. Importantly, this work builds multidisciplinary collaborations to address a serious human health concern. The proposed work will provide training in an interdisciplinary environment that is at the crossroads of microbial ecology and genetics, microbiology, hydrology, climatology, and public health. PUBLIC HEALTH RELEVANCE: Sewage contains many pathogens that can cause waterborne illness such as gastroenteritis. Human may be exposed to waterborne pathogens through contaminated drinking water or from swimming at contaminated recreational beaches. Current methods to detect fecal pollution do not distinguish the source of pollution (human or non-human), nor do they directly indicate the presence of disease-causing organisms. Our project will employ new sequencing technologies to create a comprehensive community profile of sewage derived microorganisms, from which key members will be identified that will improve our ability to detect and track sewage contamination and waterborne disease threats.